Death Receptor 5 (DR5) is a member of the TNF-superfamily of transmembrane receptors that plays a critical role in signaling the apoptotic pathway. Upregulated in cancer cells, DR5 is among the most actively pursued anti-cancer targets, both clinically and in basic research studies. However, there is great need to develop new strategies for targeting DR5 in order to maximize apoptosis in cancer cells. To do so, we need significantly more structural and biophysical data in order to understand how the receptor works. Traditionally, research has focused on the crystal structure of the extracellular, ligand-binding domain. As such, there remains a debilitating scarcity of data regarding the key structural events that occur within the transmembrane domain of the protein during transduction of the signal. Very recently, multiple high impact studies have shown that understanding the ligand-induced changes in the structure and dynamics of the transmembrane domain ?- helical dimer is the next crucial step in understanding the function of the receptor. The objective of thi application is to understand key changes in the transmembrane structure of DR5 associated with the active and inactive states of the receptor and to determine the critical amino-acid motifs that dictate changes in conformation. The rationale of this proposal is that once we understand important conformational states of the DR5 TM domain, and the most relevant motifs that stabilize states of the protein, we will be able to evaluate its potential as a therapeutic target or pharmacological regulation. Our approach combines molecular biophysics experiments on model systems (synthetic TM domains) complemented by computational simulations and molecular biology experiments on full-length receptors in living cells. We will 1) define the inter helical architecture of the DR5 transmembrane domain dimer; 2) identify key sequence alterations that either disrupt TM dimerization or stabilize alternate dimer conformations; and 3) establish the capacity to modulate TM dimer architecture and affect DR5 activation in cancer cells. The proposed research will advance understanding of TNF-Receptors in general, taking the logical but critical next steps in building a complete description of their structure-function relationship. We will create new understanding of the physical principles that dictate conformational dynamics of TM dimers, principles that are essential in a broad range of membrane protein superfamilies. In the process, we will advance the state-of-the- art in computational modeling of membrane proteins, providing a methodological roadmap for validation of models by comparison to experimental EPR spectroscopy. Working with a pancreatic cancer researcher will enable us to evaluate the TM domain of DR5 as a target for future therapeutic intervention.